Grandgenett, D. P. | Search bioRxiv

bioRxiv2025

Delineation of a novel assembly intermediate in retroviral integration pathway

Retroviral integration is mediated by viral integrase (IN) which synapses two viral long terminal repeat (LTR) DNA ends and produces a series of nucleoprotein complexes known as intasomes. While structural studies of mature intasomes have illuminated key aspects of their architecture and provided insights into the integration reaction, the sequence of events driving IN oligomerization and engagement of the viral DNA pairing remains unclear. Here, using complementary biochemical and biophysical approaches, including ensemble and single molecule FRET, we reveal that integration progresses through a key transient intermediate that leads to the mature intasome. We demonstrate that Rous Sarcoma Virus (RSV) intasome assembly pathway proceeds through a tetrameric intermediate where two IN dimers engage a single DNA end. This complex subsequently oligomerizes to form mature functional octameric intasome in which two DNA ends are juxtaposed for concerted integration. These findings provide mechanistic insights into the stepwise pathway of retroviral integration and define a previously uncharacterized intermediate critical for intasome maturation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=97 SRC="FIGDIR/small/675266v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@57a82aorg.highwire.dtl.DTLVardef@e17110org.highwire.dtl.DTLVardef@c5f4c4org.highwire.dtl.DTLVardef@4886d2_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2023

Molecular determinants for Rous sarcoma virus intasome assemblies

Integration of retroviral DNA into the host genome involves formation of integrase (IN)-DNA complexes termed intasomes. Here, we report the single-particle cryo-EM structure of the Rous sarcoma virus (RSV) strand transfer complex (STC) intasome produced with IN and a preassembled viral/target DNA substrate. The STC structure had an overall resolution of 3.36 [A] and 3 [A] in the conserved intasome core (CIC) region. Our structure demonstrated the flexibility of the distal IN subunits relative to the IN subunits in the CIC, similar to previously shown with the RSV octameric cleaved synaptic complex (CSC) intasome produced with IN and viral DNA only. An extensive analysis of higher-resolution STC structure helped in identification of nucleoprotein interactions important for intasome assembly. Using structure-function studies, we determined the mechanisms of several IN-DNA interactions critical for assembly of both RSV intasomes. We determined the role of IN residues R244, Y246 and S124 in CSC and STC intasome assemblies and their catalytic activities, demonstrating differential effects. Taken together, these studies advance our understanding of different RSV intasome structures and molecular determinants involved in their assembly.

biochemistry↗